Master

This morning the lab had a brainstorming session for potential projects I could do. The project proposal I wrote was rejected(see previous post). Here are the candidates:

1) Why is the MurE mutant hypercompetent?

MurE is an essential gene involved in the peptigoglycan pathway, and this mutant has 3 point mutations – 2 in non conserved regions and 1 in a highly conserved region. For some reason this makes it highly competent, it’s the most transformable strain we have available.

Ma and Redfield 2000. KW20 is the wild type strain, the other four are murE mutants. Transformation frequency is greatly increased in the murE mutants

We have no idea why. No one expected the cause of the phenotype to be a gene involved in cell wall synthesis. The most likely explanations have be ruled out. The cell wall isn’t any more permeable and the bacteria aren’t more sensitive to antibiotics. DNA uptake specificity is not affected. It’ a completely unexpected result that does not fit in with our current model of competence at all.

For the next few days I’ll be researching what we know about murE and learning about cell wall biosynthesis pathways to come up with some ways to approach this question. I’ll be reading E. coli papers on murE(and hoping that H.I isn’t any different), looking at old lab notebooks from different students, old microarray data, and anything else I can get a hold of.

2) Transformation in a mucous environment

How does HI transform in a more natural environment, a mucus layer? A previous student had developed a method for it so most of the tools are ready and should be very straight forwarded. I could do some interesting things, like co-cultures. However the results would be super low impact, and despite being a master in snot after completion I won’t learn any transferable skills

3) Does DNA quench competence?

Again some interest but very low impact. We have a bit of conflicted data on whether transformation is limiting or uptake, and it could be a time not quantity. The most likely answer, that cells get ‘full’ of DNA and then cant take up anymore is so boring it might not be publishable.

4) Cross-linking

This is pretty cool. Transformation is able to recombine large tracts of DNA and we have a bunch of competence genes we think are involved in protecting DNA from exonucleases.  In these mutants the transformation frequency of a marker is decreased, however we don’t know anything about the overall transformation going on. Are smaller fragments getting transformed? The reason I’d steer clear of this project is because anouther approach would be much more informative, sequencing the entire genome of transformed clones, which would not only tell us about the size of recombination tracts more exactly but also quantity. I want to work on something that will stand the test of time.

So I’ll be learning about cell walls for the next little while while all this percolates. Benchwise I’m making a rec1 clone and need to order some primers for it.

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